Nucleic Acids Research, 1995, Vol. 23, No. 9 1447-1453
© 1995
MOLECULAR BIOLOGY |
Site-specific initiation of DNA replication within the non-transcribed spacer of Physarum rDNA
Organisation Fonctionnelle du Noyau UPR 9044 CNRS, 94801 Villejuif, France
*To whom correspondence should be addressed
Received February 17, 1995. Accepted March 23, 1995.
Physarum polycephalum rRNA genes are found on extrachromosomal 60 kb linear palindromlc DNA molecules. Previous work using electron microscope visualization suggested that these molecules are duplicated from one of four potential replication origins located in the 24 kb central non-transcribed spacer [Vogt and Braun (1977) Eur. J. Blochem., 80, 557–566]. Considering the controversy on the nature of the replication origins in eukaryotic cells, where both site-specific or delocallzed Initiations have been described, we study here Physarum rDNA replication by two dimensional agarose gel electrophoresls and compare the results to those obtained by electron microscopy. Without the need of cell treatment or enrichment in replication Intermediates, we detect hybridization signals corresponding to replicating rDNA fragments throughout the cell cycle, confirming that the synthesis of rDNA molecules is not under the control of S-phase. The patterns of replication intermediates along rDNA minichromosomes are consistent with the existence of four site-specific replication origins, whose localization in the central non-transcribed spacer is in agreement with the electron microscope mapping. It Is also shown that, on a few molecules, at least two origins are active simultaneously.
+Present address: Département de Biologie, Faculté des Sciences et de Génie, Université Laval, Québec G1K 7P4, Canada
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